Transcriptional regulation of SARS-CoV-2 receptor ACE2 by SP1

Angiotensin-converting enzyme 2 (ACE2) is a major cell entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The induction of ACE2 expression may serve as a strategy by SARS-CoV-2 to facilitate its propagation. However, the regulatory mechanisms of ACE2 expression after viral infection remain largely unknown. Using 45 different luciferase reporters, the transcription factors SP1 and HNF4α were found to positively and negatively regulate ACE2 expression, respectively, at the transcriptional level in human lung epithelial cells (HPAEpiCs). SARS-CoV-2 infection increased the transcriptional activity of SP1 while inhibiting that of HNF4α. The PI3K/AKT signaling pathway, activated by SARS-CoV-2 infection, served as a crucial regulatory node, inducing ACE2 expression by enhancing SP1 phosphorylation—a marker of its activity—and reducing the nuclear localization of HNF4α. However, colchicine treatment inhibited the PI3K/AKT signaling pathway, thereby suppressing ACE2 expression. In Syrian hamsters (Mesocricetus auratus) infected with SARS-CoV-2, inhibition of SP1 by either mithramycin A or colchicine resulted in reduced viral replication and tissue injury. In summary, our study uncovers a novel function of SP1 in the regulation of ACE2 expression and identifies SP1 as a potential target to reduce SARS-CoV-2 infection.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of the coronavirus disease 2019 (COVID-19) pandemic (Wu et al., 2020;Lu et al., 2020).Patients with severe COVID-19 often exhibit pathophysiological characteristics related to a systemic inflammatory response, manifesting as acute respiratory distress syndrome and multiorgan dysfunction (Pan et al., 2021;van Eijk et al., 2021).Viral entry into host cells occurs through the binding of the SARS-CoV-2 spike protein to its cellular receptor, angiotensin-converting enzyme 2 (ACE2), followed by proteolytic cleavage of the spike protein via the transmembrane serine protease TMPRSS2 (Yan et al., 2020), facilitating the fusion of the viral and host cell membranes, a crucial step for viral internalization (Hoffmann et al., 2020).ACE2 is widely expressed in different human tissues, including the lungs, small intestine, kidneys, liver, testes, heart, and brain (Dong et al., 2020;Verdecchia et al., 2020).Such widespread expression of ACE2 may account for the multi-organ targeting observed with SARS-CoV-2.
Vaccination is regarded as an effective strategy for mitigating SARS-CoV-2 infections, as well as reducing hospitalization and mortality rates.Despite this, the evolving mutational landscape of SARS-CoV-2 has compromised the ability of certain individuals to generate a sufficient immune response through vaccination alone.Notably, the efficacy of existing vaccines has shown a marked decline against emergent variants such as Omicron (Kuhlmann et al., 2022;Wilhelm et al., 2022).Modifications in the spike protein of these variants have been observed to increase their binding affinity to ACE2, thereby enhancing transmissibility (Liu et al., 2021;McCallum et al., 2022).Consequently, need to identify and develop novel host-directed therapeutics against SARS-CoV-2 remains urgent, especially in vulnerable populations such as the elderly.
To elucidate the molecular mechanisms governing the regulation of ACE2 expression by SARS-CoV-2, we employed 45 different luciferase reporters to assay a range of signaling pathways.Our results indicated that SARS-CoV-2 up-regulated ACE2 expression by activating the transcription factor SP1, while concurrently inhibiting HNF4α via the PI3K/AKT signaling pathway.Furthermore, we demonstrated that mithramycin A (MithA), an inhibitor of SP1, exhibited efficacy against SARS-CoV-2 in both cellular and animal models.Thus, these findings suggest that SP1 serves as an important transcription factor in the regulation of ACE2 expression.

SARS-CoV-2 infection up-regulates ACE2 expression, which is inhibited by colchicine treatment
Serving as the entry receptor for SARS-CoV-2 in host cells, ACE2 is considered a promising therapeutic target against COVID-19 (Monteil et al., 2020).Consistent with prior studies indicating a significant up-regulation of ACE2 mRNA expression following SARS-CoV-2 infection (Gao et al., 2022;Wei et al., 2021;Xu et al., 2021b;Zhuang et al., 2020), we found that SARS-CoV-2 infection up-regulated ACE2 protein expression in HPAEpiCs, a human lung epithelial cell line (Figure 1A, B), with further corroboration based on immunofluorescence analysis (Figure 1C, D).Recent clinical investigations have reported a mortality benefit among COVID-19 patients treated with colchicine (Drosos et al., 2022;Elshafei et al., 2021), a drug used for the treatment of (auto-)inflammatory conditions such as acute gout and familial Mediterranean fever (Dasgeb et al., 2018;Schlesinger et al., 2020).
In the present study, we observed that colchicine treatment substantially inhibited ACE2 expression in HPAEpiCs, irrespective of SARS-CoV-2 infection status (Figure 1A-D).
As colchicine inhibits ACE2 expression, we assessed the in vitro inhibitory effects of colchicine on SARS-CoV-2 replication in HPAEpiCs.After preincubation with colchicine at different concentrations for 1 hr, cells were infected with SARS-CoV-2 for 1 hr, then cultured in fresh medium for 24 hr to measure viral RNA copy number.Based on quantitative reverse transcriptase polymerase

SARS-CoV-2 infection regulates transcriptional activities of SP1 and HNF4α
To clarify the molecular mechanisms governing ACE2 expression regulation, we used colchicine as a proof-of-principle agent.Using the Cignal Finder 45-Pathway Reporter Array (Manzini et al., 2014;Xu et al., 2021a), we analyzed a range of signaling pathways in response to SARS-CoV-2 infection, both in the presence and absence of colchicine (Figure 2A).Among the tested signaling pathways, three transcription factors (SP1, NF-κB, and GATA) enhanced by SARS-CoV-2 infection and suppressed by  colchicine treatment (Figure 2A).Conversely, two signaling pathways (HNF4α and estrogen receptor) were inactivated by SARS-CoV-2 infection but activated by colchicine treatment (Figure 2A).These transcription factors thus emerged as potential candidates for regulating ACE2 expression.To further explore this, we investigated the DNA motifs located 1.5 kb upstream of the transcription start site (TSS) of the ACE2 gene using the MEME program (Bailey et al., 2015).Our analysis revealed two significantly enriched motifs (Figure 2B), annotated as the motifs of the transcription factors SP1 (p=6.1e-5) and HNF4α (p=4.2e-5).These findings suggest that these two transcription factors are likely regulators of ACE2 expression.
To assess the impact of SARS-CoV-2 and colchicine on SP1 and HNF4α activities, we examined their subcellular distributions using immunofluorescence analysis.Although SP1 was mainly located in the nucleus of HPAEpiCs (Figure 2-figure supplement 1), only a small proportion of total SP1 was phosphorylated at Thr453 (Figure 2C), a modification indicative of its activation (Milanini-Mongiat et al., 2002).SARS-CoV-2 infection markedly increased the phosphorylation levels of SP1 at Thr453, whereas colchicine treatment significantly inhibited the phosphorylation of SP1, regardless of the presence or absence of SARS-CoV-2 infection (Figure 2C, D).In contrast to SP1, HNF4α displayed both nuclear and cytoplasmic distribution in HPAEpiCs under basal conditions (Figure 2E).SARS-CoV-2 infection prompted a redistribution of HNF4α from the nucleus to cytoplasm, while colchicine treatment induced its nuclear accumulation both in the presence and absence of SARS-CoV-2 infection (Figure 2E, F).These results suggest that SARS-CoV-2 infection activates SP1 and inactivates HNF4α, which can be counteracted by colchicine treatment.

Involvement of SP1 and HNF4α in ACE2 expression
Next, we investigated whether colchicine inhibited ACE2 expression via regulation of SP1 and HNF4α.First, western blot analysis demonstrated that treatment with MithA, a selective SP1 inhibitor, or knockdown of SP1 by small interfering RNA (siRNA) down-regulated ACE2 protein expression in HPAEpiCs (Figure 3A, B).However, inhibition of SP1 by MithA or siSP1 did not further reduce the down-regulation of ACE2 protein levels achieved by colchicine.In contrast, treatment with the HNF4α antagonist BI6015 and knockdown of HNF4α by siRNA up-regulated ACE2 protein expression (Figure 3C, D), which was blocked by colchicine administration.
Immunofluorescence staining further confirmed that although MithA treatment inhibited the protein expression of ACE2, it did not further reduce the expression of ACE2 suppressed by colchicine (Figure 3E, F).Supplementation with BI6015 markedly increased the expression of ACE2 in HPAEpiCs, which was reduced by colchicine treatment (Figure 3E, F).Similar results were obtained in human epithelial cells A549, human renal tubular cells HK-2, and human hepatoma cells Huh-7 (Figure 3-figure supplements 1 and 2).
Based on a luciferase reporter gene containing the ACE2 promoter, MithA treatment inhibited luciferase activity, but not in the presence of colchicine (Figure 3G), while BI6015 treatment increased luciferase activity.In addition, chromatin immunoprecipitation (ChIP)-qPCR analysis indicated that the binding of SP1 to the GC box of the ACE2 promoter was significantly reduced, while the binding of HNF4α to the AGGTCA element was markedly increased after colchicine treatment (Figure 3H).As MithA inhibited the expression of ACE2, we also tested its effects on SARS-CoV-2 infection in vitro.Results showed that MithA inhibited SARS-CoV-2 replication in HPAEpiCs, with an EC 50 value of 0.1948 μM (Figure 3-figure supplement 3).These data suggest that SP1 and HNF4α exert opposing effects on the transcriptional regulation of ACE2 expression, thereby influencing cellular susceptibility to SARS-CoV-2 infection.

Dual antagonism of SP1 and HNF4α
Interestingly, treatment with the SP1 inhibitor MithA markedly suppressed SP1 phosphorylation, whereas treatment with the HNF4α antagonist BI6015 increased SP1 phosphorylation at Thr453 (Figure 4A, B).In addition, BI6015 induced the cytoplasmic translocation of HNF4α, while MithA promoted its nuclear accumulation (Figure 4C, D).Western blot analysis showed an elevation in SP1 phosphorylation levels following the knockdown of HNF4α by siRNA (Figure 4E, F), whereas the knockdown of SP1 had no discernible impact on the total levels of HNF4α.We subsequently confirmed potential interactions between SP1 and HNF4α in HPAEpiCs using co-immunoprecipitation
The PI3K/AKT signaling pathway plays an important role in regulating the transcriptional activities of SP1 and HNF4α (Adapala et al., 2019;Gómez-Villafuertes et al., 2015;Li et al., 2019;Zhao et al., 2015).Activation of AKT promotes the stability and localization of SP1 by phosphorylating SP1 at Thr453 and Thr739 (Adapala et al., 2019;Gómez-Villafuertes et al., 2015;Zhao et al., 2015), and prevents the nuclear translocation of HNF4α (Li et al., 2019).Consistent with these observations, we found that suppression of AKT by siRNA or its inhibitors significantly reduced the accumulation of phospho-SP1 in the nucleus, but markedly promoted the nuclear translocation of HNF4α (Figure 5H-K).Taken together, these findings suggest that the down-regulation of ACE2 expression by colchicine is contingent upon the inhibition of the PI3K/AKT signaling pathway, which, in turn, modulates the transcriptional activities of SP1 and HNF4α.

Inhibition of SP1 reduces viral load and damage to the respiratory and renal systems
We tested whether inhibition of SP1 by colchicine and MithA inhibits the replication of SARS-CoV-2 in vivo using Syrian hamsters (Mesocricetus auratus), an animal model used for the study of COVID-19 pneumonia and therapeutic evaluation (Chan et al., 2020;Choudhary et al., 2022;Muñoz-Fontela et al., 2020;Rosenke et al., 2021;Sia et al., 2020).These hamsters were intranasally infected with SARS-CoV-2 at a 50% tissue culture infectious dose (TCID 50 ) of 10 4 .After 1 hr, the hamsters were inoculated intraperitoneally with colchicine or MithA at 0.2 mg/kg, respectively.A mock group was treated with vehicle (1% DMSO and 99% saline) using the same route and timing (Figure 6-figure supplement 1).The animals were dosed every 24 hr with either colchicine or MithA at 0.2 mg/kg, respectively.Lung and tracheal samples were collected at 3 days post-infection (dpi) to assess viral RNA and ACE2 expression.Immunofluorescence analysis demonstrated that the expression of ACE2 was Source data 7. Original file for ChIP analysis in Figure 3H.Subsequent histopathological analysis of lung tissues 3 dpi revealed a range of pulmonary abnormalities, including bronchial epithelial cell necrosis and nuclear pyknosis, extensive alveolar hemorrhage, marked infiltration of inflammatory cells, significant edema, and notably thickened alveolar walls (Figure 6E, G).Although similar pathological features were present, their severity was markedly reduced in the treatment groups compared to the mock group.Using Masson's trichrome staining, collagen deposition indicative of fibrosis was evident in the lungs of infected hamsters (Figure 6F, H).Of note, treatment with either colchicine or MithA effectively reversed lung fibrosis.These findings suggest that colchicine and MithA antagonize SARS-CoV-2 replication in the lung and trachea while also ameliorating associated histopathological damage.
Although acute kidney injury associated with severe SARS-CoV-2 serves as an independent risk factor for in-hospital death in patients (Nadim et al., 2020), whether SARS-CoV-2 directly infects the kidney remains unclear (Smith and Akilesh, 2021;Wysocki et al., 2021).Prevailing evidence appears to favor indirect kidney injury by SARS-CoV-2 (Smith and Akilesh, 2021).Here, immunofluorescence analysis detected the substantial presence of SARS-CoV-2 in the kidneys of infected hamsters, an effect markedly mitigated by colchicine or MithA treatment (Figure 7A, B).Further analysis also showed that both drugs effectively down-regulated ACE2 expression (Figure 7C, D).Histopathological evaluation showed significant renal damage in SARS-CoV-2-infected hamsters, including renal tubular epithelial cell nuclear pyknosis, brush border disappearance, renal interstitial vascular congestion, inflammatory cell infiltration, and glomerular atrophy (Figure 7E, F).In the drug-treated hamsters, however, the glomeruli displayed uniform distribution and intact structure, tubular epithelial cells appeared normal, and brush borders showed neat arrangement, with no evident abnormality in the renal medulla or obvious hyperplasia in the renal interstitium.Collectively, these findings underscore the therapeutic efficacy of colchicine and MithA in ameliorating renal histopathological alterations.

Discussion
Based on our study and current understanding of SARS-CoV-2 infection, a mechanistic model is proposed regarding how SARS-CoV-2 infection induces ACE2 expression via two transcription factors, SP1 and HNF4α, in host cells.Under physiological conditions, SP1 is primarily localized in the nucleus, while HNF4α exhibits a dual distribution in the nucleus and cytoplasm.These transcription factors exert opposing effects on the regulation of ACE2 expression, thereby maintaining basal expression of ACE2.Upon infection by SARS-CoV-2, the PI3K/AKT signaling pathway becomes activated (Klann et al., 2020).This activation subsequently promotes the transcriptional activity of SP1 through increased phosphorylation in the nucleus and suppresses the transcriptional activity of HNF4α by inducing its translocation to the cytoplasm.Disruption of this balance leads to the up-regulation of ACE2 after viral infection.Given that ACE2 serves as the entry receptor for SARS-CoV-2, induction of ACE2 expression may represent a strategic adaptation of the virus to facilitate its own propagation.
Recent advances in multi-omics approaches have facilitated our understanding of host responses to SARS-CoV-2, thereby accelerating the development and repositioning of therapeutic agents against COVID-19 (Chu et al., 2021;Ho et al., 2021;Kamel et al., 2021;Klann et al., 2020;Lu et al., 2022).Phosphoproteomic analysis of SARS-CoV-2-infected cells has revealed the activation of the growth factor receptor (GFR) and its downstream pathways, including the RAF/MEK/ERK MAPK and PI3K/AKT/mTOR signaling pathways, in SARS-CoV-2-infected cells (Klann et al., 2020).Blockage of the GFR signaling pathway via application of prominent anti-cancer drugs, such as sorafenib (RAF inhibitor), RO5126766 (dual RAF/MEK inhibitor), pictilisib (PI3K inhibitor), and omipalisib (dual PI3K and mTOR inhibitor), effectively prevents SARS-CoV-2 replication in cellular models.Here, through screening 45 signaling pathways in SARS-CoV-2-infected HPAEpiCs, either in the presence or absence of colchicine, our study elucidated the opposite regulatory roles of two transcription factors, SP1 and HNF4α, in the modulation of ACE2 expression at the transcriptional level, which exerted positive and        negative regulatory effects, respectively.Notably, our findings indicated that the PI3K/AKT signaling cascade, activated following SARS-CoV-2 infection (Klann et al., 2020), served as a crucial regulatory node in the induction of ACE2 expression by enhancing and reducing the transcriptional activities of SP1 and HNF4α, respectively.
Our findings also showed that inhibition of SP1 activity, leading to the down-regulation of ACE2 expression, effectively mitigated SARS-CoV-2 replication in the lung and trachea of Syrian hamsters.Importantly, this decrease in viral replication was associated with a marked reduction in lung pathology.In addition to lung-associated issues, acute kidney injury has also been identified as an extrapulmonary manifestation of severe COVID-19 (Legrand et al., 2021), although the direct causal association between SARS-CoV-2 infection and acute kidney injury remains controversial (Legrand et al., 2021;Smith and Akilesh, 2021).Recent evidence has indicated that SARS-CoV-2 can directly infect kidney cells in human-induced pluripotent stem cell-derived kidney organoids (Jansen et al., 2022).Consistent with these findings, we found that colchicine treatment effectively suppressed ACE2 expression in the proximal kidney tubule, which was associated with diminished SARS-CoV-2-induced kidney injury in Syrian hamsters.These results support ACE2 as a promising therapeutic target against COVID-19 (Monteil et al., 2020).Notably, proxalutamide, a potent androgen receptor antagonist, has been shown to suppress androgen-induced ACE2 expression (Qiao et al., 2021).Moreover, RIPK1 inhibition has been found to reduce viral loads in SARS-CoV-2-infected human lung organoids, accompanied by a down-regulation in the transcriptional induction of ACE2 (Xu et al., 2021a).A recent study also demonstrated that inhibition of FXR activity via UDCA can lead to the down-regulation of ACE2 expression, consequently reducing SARS-CoV-2 infection in vitro, in vivo, and ex vivo (Brevini et al., 2023).
In further examination of ACE2 expression, an unexpected observation emerged.Notably, inhibition of SP1 by its inhibitor MithA induced nuclear accumulation of HNF4α, whereas inhibition of HNF4α by its antagonist BI6015 increased phosphorylation levels of SP1 in the nucleus.Further exploration is warranted to elucidate the mechanisms underlying the antagonistic interactions between the two transcription factors.Although the established role of MithA is to disrupt the binding of SP1 to its consensus site (Lee et al., 2011), it is also capable of inducing proteasome-dependent SP1 degradation (Choi et al., 2014;Lee et al., 2012).This may explain why MithA treatment significantly suppressed the phosphorylation of SP1 in our study.Similarly, BI6015, known to repress the DNAbinding activity HNF4α (Kiselyuk et al., 2012), can also diminish HNF4α expression.This may provide a ready explanation of our observation that BI6015 could reduce nuclear accumulation of HNF4α induced by colchicine.Our results demonstrated that non-phosphorylated SP1 interacted with HNF4α and potentially inhibited the phosphorylation of SP1.Notably, upon reduction of HNF4α protein levels by BI6015, SP1 was released and subsequently phosphorylated by AKT, resulting in the binding of phosphorylated SP1 to the GC box and subsequent up-regulation of ACE2 expression.Conversely, upon reduction of SP1 protein levels by MithA, HNF4α was released, which inhibited ACE2 expression by binding to the HNF4α-specific binding motif.These observations enhance our understanding of the counteracting transcriptional activities between SP1 and HNF4α.However, additional research is needed to elucidate these mechanisms, particularly in the context of current findings.
This study has several limitations.Firstly, although SP1 was identified as a pivotal transcription factor in modulating ACE2 expression via the action of colchicine and MithA, neither of these compounds currently qualify as a candidate for the treatment of COVID-19.Primarily used to treat several types of cancer, including testicular cancer (Kennedy and Torkelson, 1995) and myeloid leukemia (Dutcher et al., 1997), the clinical application of MithA is substantially limited by its range of adverse effects, including liver, kidney, gastrointestinal, and bone marrow toxicity (Green and Donehower, 1984;Kennedy, 1970;Quarni et al., 2019).Additionally, the efficacy of colchicine as a treatment for COVID-19 remains inconclusive.While some studies suggest benefits (Chiu et al., 2021;Drosos et al., 2022;Elshafei et al., 2021), others indicate negligible impact on mortality or disease progression (RECOVERY Collaborative Group, 2021;Mikolajewska et al., 2021).Secondly, although inhibition of SP1 phosphorylation in Syrian hamsters was shown to effectively reduce viral replication and inflammatory responses, these effects need to be further evaluated in other model animals, such as ferrets and non-human primates (Shi et al., 2020).
Increased ACE2 expression in respiratory and pulmonary tissues has been implicated in the exacerbation of COVID-19 symptom severity among elderly individuals (Inde et al., 2021).Importantly, the Omicron variant of SARS-CoV-2, the current dominant global strain, exhibits a spike protein with a six-to nine-fold enhanced binding affinity for ACE2 (Yin et al., 2022).Thus, our observations have the potential to inform the development of novel host-targeting strategies to combat COVID-19, particularly in elderly populations affected by the SARS-CoV-2 Omicron variant.

SARS-CoV-2
The SARS-CoV-2 strain (accession number: NMDCN0000HUI) was provided by the Guangdong Provincial Center for Disease Control and Prevention (Guangzhou, China).The virus was propagated in African green monkey kidney epithelial cells (Vero-E6) (ATCC, No. 1586) and titrated.All infection experiments were performed in a Biosafety Level-3 (BLS-3) Laboratory.

Half-maximal effect concentration (EC 50 )
The HPAEpiCs were seeded at a density of 1.6×10 4 cells/well in 48-well plates and grown overnight at 37 °C.The cells were infected with SARS-CoV-2 at an MOI of 1 and the test compounds were added to the wells at different concentrations.After 1 hr of incubation at 37 °C, the virus-drug mixture was removed and washed three times with phosphate-buffered saline (PBS) to eliminate free virus, before being replenished with fresh medium containing the compounds.After 48 hr, the supernatants were collected to extract viral RNA for RT-qPCR analysis.The EC 50 values were calculated using a doseresponse model in GraphPad Prism v8.0 (GraphPad Software Inc, La Jolla, CA, USA).

Cellular antiviral activity assay
The HPAEpiCs were seeded at a density of 4×10 5 cells/well in 24-well plates and grown overnight at 37 °C.Following preincubation with the test compounds for 2 hr, the cells were infected with SARS-CoV-2 at an MOI of 1.After 1 hr of incubation at 37 °C, the virus-drug mixtures were replaced with fresh medium containing compounds.After 24 hr, the cells were collected to extract total RNA and total cell protein.Viral RNA was quantified using a Thunderbird Probe One-step RT-qPCR Kit (QRZ-101, Toyobo, Shanghai, China).The TaqMan primers used for SARS-CoV-2 were 5'-GGG GAA CTT CTC CTG CTA GAA T-3' and 5'-CAG ACA TTT TGC TCT CAA GCT G-3' with SARS-CoV-2 probe FAM-TTG CTG CTG CTT GAC AGA TT-TAMRA-3'.

45-Pathway Reporter Array
Cignal Finder 45-Pathway Reporter Arrays (CCA-901, Qiagen, Hilden, Germany) were used according to the manufacturer's instructions to identify potential pathways regulated by SARS-CoV-2.Briefly, HPAEpiCs were reverse transfected with firefly luciferase reporter constructs containing response elements for the indicated pathways, and with control Renilla luciferase constructs for 24 hr.Cells were then pretreated with colchicine for 2 hr and incubated with SARS-CoV-2 for 24 hr.Luciferase activities of the cells were then measured using a dual-luciferase reporter assay system (E1910, Promega) on a fluorescent microplate reader (Molecular Devices Inc).Reporter luciferase activity was normalized to Renilla luciferase activity for each sample.All experiments were performed with three biological replicates.

Luciferase reporter assay
The HPAEpiCs were co-transfected at a density of 3×10 3 with pRL-SV40 vector and phACE2-promoter-TA-luc (D2488, Beyotime) using Lipofectamine 3000 reagent (L3000015, Invitrogen).After 48 hr of transfection, luciferase activity was measured using the dual-luciferase reporter assay system (E1910, Promega, Shanghai, China) on a fluorescent microplate reader (Molecular Devices Inc, Sunnyvale, CA, USA).The ratio of firefly luciferase to Renilla luciferase was calculated for each experiment and averaged from three replicates.All experiments were performed with three biological replicates.

Transcription factor binding motif enrichment
The ACE2 promoter sequence (1 500 bp upstream of TSS) was extracted from the NCBI database.The DNA-binding motifs of transcription factors estrogen receptor GATA6, HNF4α, NF-κB, and SP1 were obtained from the JASPAR CORE database (genereg.net).MEME Suite (meme-suite.org)was used to determine the enrichment of the transcription factor motifs and binding sites in the ACE2 promoter sequence (Bailey et al., 2015).FIMO analysis was performed using stringent criteria, including p<1E-4 and a maximum of two mismatched residues.

ChIP-qPCR
ChIP was performed as described previously (Tao et al., 2016) using a ChIP assay kit (P2078, Beyotime) following the manufacturer's directions.After crosslinking with formaldehyde, the chromatin solutions were sonicated and incubated with anti-SP1 (9389, 1:100, Cell Signaling Technology) and anti-HNF4α (ab181604, 1:100, Abcam) antibodies and control IgG, then rotated overnight at 4 °C, respectively.After purification using a DNA purification kit (BioTeke Corp.), the immunoprecipitated DNA was detected for PCR analysis.All ChIP-qPCR experiments were performed with three biological replicates.

Co-immunoprecipitation (co-IP)
For the co-IP experiments, HPAEpiCs were lysed on ice for 30 min in cell lysis buffer (P0013, Beyotime, Shanghai, China).After centrifugation at 12,000 rpm for 30 min at 4 °C, the supernatant was collected and incubated with anti-HNF4α antibodies (ab181604, 1:70, Abcam, Shanghai, China) overnight.After 4 hr of incubation with Protein A Agarose (20333, Thermo Scientific, Shanghai, China) at 4 °C, the complexes were washed three times.Immunoblotting was performed after elution.

Hematoxylin and eosin (H&E) staining and histopathology scores
Lung, trachea, and kidney samples were collected from the Syrian hamsters, then fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned (4 μm thick).The tissue sections were then stained with H&E for histopathological examination.After staining, a four-point scoring system was applied to assess the severity of pathology in tissues, evaluated by a trained pathologist (L.-Q.W.) blind to group identity.Scoring was graded from 0, indicating no pathological change, to 1-4, indicating increasing severity.Lung histopathological scores were assessed based on alveolar wall thickening, edema, hemorrhage, and inflammatory cell infiltration.Kidney histopathological scores were assessed based on cellular degeneration, necrosis, hemorrhage, inflammatory cell infiltration, and congestion.Histopathological scores represented the sum of the injury subtype scores for each condition on a 0-20 scale.

Statistical analysis
Differences in gene expression, mRNA and protein levels, viral RNA, Luciferase reporter assay, ChIP-qPCR assay, and fluorescence intensity were assessed by Student's t-test.Data were analyzed using GraphPad Prism v8 (GraphPad Software Inc, La Jolla, CA, USA).The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Figure 1 .
Figure 1.SARS-CoV-2 infection up-regulated ACE2 expression, which was suppressed by colchicine.(A and B) SARS-CoV-2 infection up-regulated protein levels of ACE2.Colchicine (20 nM) significantly reduced protein levels of ACE2 in HPAEpiCs.Blot is typical of three independent experiments (A).Quantification of ACE2 to Actin ratio (B).Results are means ± standard deviation (SD) of three independent experiments.*p<0.05(Student's t-test).(C) Representative images of immunofluorescence staining for ACE2.Scale bar: 10 μm.(D) Quantification of ACE2 fluorescence intensity.Results are means ± SD of three independent experiments.*p<0.05(Student's t-test).Veh, Vehicle.Col, Colchicine.nCoV-2, SARS-CoV-2.The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1.Original uncropped western blot images in Figure 1A (anti-ACE2 and anti-Actin).Source data 2. PDF containing Figure 1A and original scans of relevant western blots (anti-ACE2 and anti-Actin) with highlighted bands and sample labels.Source data 3. Original file for quantification of ACE2 to Actin ratio in Figure 1B (anti-ACE2 and anti-Actin).Source data 4. Original file for quantification of ACE2 fluorescence intensity in Figure 1D.

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Figure supplement 1-source data 1.Original file for determination of viral load in Figure 1-figure supplement 1A.

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Figure supplement 1-source data 2. Original file for dose-response analysis in Figure 1-figure supplement 1B.

Figure supplement 1 .
Figure supplement 1. SP1 was primarily located in nucleus of HPAEpiCs.

Figure 3
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Figure supplement 1 .
Figure supplement 1. Immunofluorescence analysis of expression and localization of ACE2 in A549 cells.

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Figure supplement 1-source data 1.Original file for quantification of ACE2 fluorescence intensity in Figure 3-figure supplement 1B.

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Figure supplement 2-source data 3. PDF containing Figure 3-figure supplement 2A, C and original scans of relevant western blot analysis (anti-ACE2 and anti-Actin) with highlighted bands and sample labels.

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Figure supplement 2-source data 4. Original file for quantification of ACE2 to Actin ratio in Figure 3-figure supplement 2B, D (anti-ACE2 and anti-Actin).

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Figure supplement 2-source data 5. Original file for determination of viral load in Figure 3-figure supplement 2E, F.

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Figure supplement 3-source data 1.Original file for dose-response analysis in Figure 3-figure supplement 3.

Figure 4 .
Figure 4. SP1 and HNF4α antagonized each other via protein-protein interactions.(A and B) Supplementation with colchicine (20 nM) and MithA (100 nM) significantly suppressed SP1 phosphorylation (p-SP1) in HPAEpiCs, which was reversed by treatment with BI6015 (20 μM).Representative images of immunofluorescence staining for p-SP1 (A).Scale bar: 10 μm.Quantification of p-SP1 fluorescence intensity (B).Results are means ± SD of three independent experiments.*p<0.05,**p<0.01(Student's t-test).(C and D) Supplementation with colchicine (20 nM) and MithA (100 nM) promoted nuclear accumulation of HNF4α in HPAEpiCs, which was inhibited by treatment with BI6015 (20 μM).Representative images of immunofluorescence staining for HNF4α (C).Scale bar: 10 μm.Quantification of HNF4α fluorescence intensity (D).Results are means ± SD of three independent experiments.*p<0.05,**p<0.01(Student's t-test).(E and F) Phosphorylation levels of SP1 and total protein levels of HNF4α were measured in HPAEpiCs by western blotting.Blot is typical of three independent experiments (E).Quantification of p-SP1 or HNF4α to Actin ratio (F).Results are means ± SD of three independent experiments.*p<0.05,ns, not significant (Student's t-test).(G) Interactions between SP1 and HNF4α were measured by co-IP in HPAEpiCs.NC, Negative control.Veh, Vehicle.Col, Colchicine.The online version of this article includes the following source data for figure 4: Source data 1.Original file for quantification of p-SP1 fluorescence intensity in Figure 4B.Source data 2. Original file for quantification of HNF4α fluorescence intensity in Figure 4D.Source data 3. Original uncropped western blot images in Figure 4E (anti-p-SP1, anti-HNF4α, and anti-Actin).Source data 4. PDF containing Figure 4E, G and original scans of relevant western blot analysis (anti-p-SP1, anti-HNF4α, and anti-Actin) with highlighted bands and sample labels.Source data 5. Original file for quantification of p-SP1 or HNF4α to Actin ratio in Figure 4F (anti-p-SP1, anti-HNF4α, and anti-Actin).Source data 6.Original uncropped western blot images in Figure 4G.

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Figure supplement 1
Figure supplement 1-source data 1.Original file for dose-response analysis in Figure 5-figure supplement 1A, B.

Figure supplement 1 .
Figure supplement 1.Schematic representation of experimental design for SARS-CoV-2 infection in Syrian hamsters.

Figure supplement 2 .
Figure supplement 2. Colchicine and MithA blocked replication of SARS-CoV-2 by inhibiting ACE2 expression in both lung and trachea of hamsters.

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Figure supplement 2-source data 1.Original file for quantification of ACE2 fluorescence intensity in Figure 6-figure supplement 2A, B.

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Figure supplement 2-source data 2. Original file for quantification of SARS-CoV-2-N fluorescence intensity in Figure 6-figure supplement 2C, D.

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Figure supplement 2-source data 3. Original file for quantification of p-SP1 fluorescence intensity in Figure 6-figure supplement 2E, F.

Figure 7 .
Figure 7. Inhibition of SP1 inhibited SARS-CoV-2 replication and reduced kidney pathology in Syrian hamsters.(A and B) Treatment with colchicine or MithA inhibited SARS-CoV-2 replication in kidney of hamsters.Representative images of immunofluorescence staining of SARS-CoV-2-N in kidney of hamsters (A).Parts on right side are high-power images.Scale bar: 20 μm.Quantification of SARS-CoV-2-N fluorescence intensity in kidney of hamsters (B).Error bars show means ± SD. **p<0.01,***p<0.001(Student's t-test).(C and D) Treatment with colchicine or MithA inhibited ACE2 expression in kidney of hamsters infected with SARS-CoV-2.Representative images of immunofluorescence staining of ACE2 in kidney of hamsters (C).Parts on right side are high-power images.Scale bar: 20 μm.Quantification of ACE2 fluorescence intensity in kidney of hamsters (D).Error bars show means ± SD. *p<0.05,**p<0.01(Student's t-test).(E and F) Supplementation with colchicine or MithA attenuated histopathological damage in kidney of hamsters infected with SARS-CoV-2.Representative images of H&E staining in kidney of hamsters infected with SARS-CoV-2 at 3 dpi (E).Histopathology of kidney showed renal interstitial vascular congestion (black arrow), renal tubular epithelial cell nuclear pyknosis (red arrow), brush border disappearance (green arrow), renal interstitial inflammatory cell infiltration (blue arrow), and glomerular atrophy (yellow arrow).Parts on lower side are high-power images of red, green, and blue arrows, respectively.Scale bar: 40 μm.Summary of kidney lesion scoring in different groups at 3 dpi (n=5 each group) (F).Error bars show means ± SD. *p<0.05,**p<0.01(Student's t-test).Ctrl, Control.Veh, Vehicle.Col, Colchicine.nCoV-2, SARS-CoV-2.The online version of this article includes the following source data for figure 7: Source data 1.Original file for quantification of SARS-CoV-2-N fluorescence intensity in Figure 7B.Source data 2. Original file for quantification of ACE2 fluorescence intensity in Figure 7D.Source data 3. Original file for lesion scores in Figure 7F.
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